ORAL CYTOPATHOLOGICAL

CHANGES IN HABITUAL SNUFF

(NASWAR) USERSMohyman Sarfraz, A.H. Nagi

Department of Oral Pathology

University of Health Sciences,

Lahore, Pakistan

INTRODUCTION

• Dipping tobacco, traditionally known as moist/wetsnuff, is a type of finely ground or shredded,moistened smokeless tobacco product (1).

• It is commonly known by various terms such as dip• It is commonly known by various terms such as dipand rubs or chews, also known as naswar in nativelanguage of Urdu. The act of using it is calleddipping, packing or more specifically packing a lip(2).

1. Axell, T., Mornstad, H. and Sundstorm, B., 1976. The relation of the clinical picture to the histopathology and histochemical study on snuff dipper’s lesion in a Swedish population. J. Oral Pathol., 5:229-236.

2. Asma, S., Backinger, C., Blatt, B., Rosaria, M., Grana, R., Ricardo, G., Prakash, G., Ali, I., Brian, J., Michelle, R. and Susan G.R., 2002. Smokeless Tobacco Fact Sheets. 3rd International Conference onSmokeless Tobacco. Advancing Science & Protecting Public Health. Stockholm, Sweden.

• Wet snuff, also known as dipping tobacco, is made fromfresh tobacco leaves, calcium oxide, wood ash, colouringand flavouring agents. Wet snuff is held in the mouth for10 to 15 minutes and is not chewed, thus people take iton hourly basis.

Kazmi, S.Y., 2010. Snuff : Tobacco of the Pashtuns, Karachi, 2010. [online] Available at:< http://www.demotix.com/news/272599/snuff-tobacco-pashtuns-karachi>

(Accessed on 13 March 2012).

• The smokeless tobacco products which carry

specific mutagenicity are toombak used in

Sudan, shamma in Saudi Arabia while

powdered tobacco and alkali mixtures such as

naswar are used in Central Asia and Pakistan.naswar are used in Central Asia and Pakistan.

• Warnakulasuriya, S., Sutherland, G. and Scully, C., 2005. Tobacco, oral cancer, and treatment of dependence. Oral Oncology, 41: 244-260.

• A number of studies from United States, South Africa and Scandinivia have confirmed that the lesions in the oral mucosa appear in the anatomical location where the snuff is placed.

• Greer, R.O., Poulson, T.C. and Boone, M.E., 1986. Smokeless tobacco-associated oral changes in juvenile, adult and geriatric patients. Clinical and

histomorphological features. Gerodontic, 2: 87-98.

• In a retrospective Swedish study, 41 cases ofcancer occurred on the anterior vestibular partof the oral cavity in male snuff dippers and in 29of the oral cavity in male snuff dippers and in 29cases (71 %) the tumor had arisen in the exactlocation where the snuff had been placed.

• Sundstorm, B., Mornstad, H. and Axell, T., 1982. Oral carcinomas associated with snuff dipping, some clinical and histological characteristics of 23

tumors in Swedish male. J. Oral Pathol., 11: 245-251.

• The easy availability, low cost and lack ofawareness of its negative impact on healthincreases its consumption.

OBJECTIVE

• To observe the cytopathological changes in oralmucosa of habitual wet snuff users*.mucosa of habitual wet snuff users*.

*The operational definition of habitual wet snuff users refers to people who have been using naswar (wet snuff) regularly for more then 1 year.

MATERIAL AND METHODS

STUDY DESIGN:This was an observational, descriptive study.

SETTING:The study was conducted in the Dept. of MorbidAnatomy and Histopathology at University ofAnatomy and Histopathology at University ofHealth Sciences, Lahore, Pakistan

DURATION OF STUDY:The study was completed in eight months (FEB –SEPT 2013) after approval of synopsis by AdvanceStudies and Research Board of UHS, Lahore,Pakistan.

SAMPLE SIZE:

The calculated sample size was

100

(Calculated by P.A.S.S 2008)1

SAMPLE TECHNIQUE:

Non-probability,

Convenient sampling

SAMPLE SELECTION:

Inclusion Criteria

• Habitual snuff users of minimum age15 years

• Minimal duration of usage 1 year

• Males only

Exclusion Criteria

• Inadequate smear

• Cigarette smokers• Cigarette smokers

• Cases who are using other forms ofsmokeless tobacco like paan, betel nut,quid, chalia etc

• Patients having associated debilitatingco-morbid conditions

• Patients with immune disorders.

• Patients who are undergoingtreatment/therapy for alreadydiagnosed lesions

Guenther, W.C. (1977). Desk calculation of probabilities for the distribution of the sample correlation coefficient. Am Statis. Vol. 31, No. 1, pp. 45-48.

Clinical Details:

• Socio-demographic information (name, age,occupation, full address, family/medical history)occupation, full address, family/medical history)was obtained along with relevant clinicalinformation.

• All the information was collected on speciallydesigned proformas and recorded.

• After informed consent oral cytological smearswere acquried from 100 patients randomly fromdifferent areas within the city of Lahore,Pakistan.

• Smears were prepared from oral mucosascrapings and examined microscopically usingH/E and special stains including Papanicolou,Gomori Methenamine Silver and Giemsa stain.

RESULTS

• The mean age of the total n = 100 patients was

42.5 + 13.8 years

• Age range being 15 to 70 years

• Most of the cases (97 %) belonged to low

socioeconomic status

• The oral hygiene was found to be poor in 91 %,• The oral hygiene was found to be poor in 91 %,

fair in 8 % and good in 1 % only.

• The patients were classified into two groups

according to age�Group 1<30 years (mean age = 20.2 years)

�Group 2> 30 years (mean age = 48.5 years)

43%

57%

40%

50%

60%

AGE GROUPS

AGE GROUPS

Group 1 < 30 years of age 43 %

Group 2 > 30 years of age 57 %

0%

10%

20%

30%

Group 1 Group 2

CLINICAL FEATURES OBSERVED IN n = 100

PATIENTS

35%

11%

4%

4%

percentages

25%

21%

keratosis 100 %

erythroplakia 73%

gingivitis 62 %

white plague 31 %

oral pigmentation 12 %

mucositis 12 %

WRINKLED MUCOSA

GINGIVITIS AND PERIODONTITIS

WHITE LESION

TOBACCO POUCH KERATOSIS

PIGMENTATION

ASSOCIATIONS OF AGE GROUPS WITH

CLINICAL FEATURES

• When both the age groups of patients werecompared with the clinical features, thecompared with the clinical features, thefollowing associations were observed.

Group 1n (% )

Group 2n (%)

Totaln (%)

P Value

MUCOSITIS 43 ( 100 ) 57 (100) 100 (100) --

ULCERATION 43 (100 ) 57 (100 ) 100 (100) --

WHITE PLAQUE 10 ( 23.3) 21 ( 37 ) 31 (31) 0.146

XEROSTOMIA 43 (100 ) 57 (100) 100 (100 ) --

ERYTHROPLAKIA 32 ( 74.4 ) 41 (72 ) 73 (73) 0.781

*Group 1 = age group less then 30 years

*Group 2 = age group above 30 years

ERYTHROPLAKIA 32 ( 74.4 ) 41 (72 ) 73 (73) 0.781

PERIODONTITIS 18 (42 ) 17 ( 30) 35 (35) 0.212

TOBACCO POUCH KERATOSIS

43 (100 ) 57 (100) 100 (100) --

GINGIVITS 26 (61) 36 (62 ) 62 (62 ) 0.784

• When the patients were classified according toduration of naswar usage the maximum numbern = 54 was seen in the group 1 followed by n = 41and n = 5 in group 2 and group 3 respectively.

• When the duration of usage was associated withthe clinical observations, the followingassociations were recorded

• *Group 1 = mild � less then 5 years of intake

*Group 2 = moderate � between 5 to 10 years of usage

*Group 3 = heavy � more then 10 years of usage

Group 1 n ( %)

Group 2n (%)

Group 3n (%)

Total P value

MUCOSITIS 54 (100 ) 41 (100) 5 (100) 100 (100) --

ULCERATION 54 (100) 41 (100 ) 5 (100) 100 (100) --

WHITE PLAQUE 14 (26 ) 15 (37 ) 2 (40 ) 31 (31 ) 0.503

XEROSTOMIA 54 (100 ) 41 ( 100 ) 5 (100 ) 100 (100) --

ERYTHROPLAKIA 40 (74.1 ) 31 (76) 2 ( 40 ) 73 (73) 0.254

PERIODONTITIS 23 (43 ) 12 (29.3) 0 ( 0 ) 35 ( 35) 0.121

*Group 1 = mild � less then 5 years of intake

*Group 2 = moderate � between 5 to 10 years of usage

*Group 3 = heavy � more then 10 years of usage

PERIODONTITIS 23 (43 ) 12 (29.3) 0 ( 0 ) 35 ( 35) 0.121

TOBACCO POUCH KERATOSIS

54 ( 100 ) 41 (100 ) 5 (100) 100 (100 ) --

GINGIVITS 32 (59.3 ) 26 (63.4 ) 4 ( 80 ) 62 ( 62 ) 0.738

• When the patients were grouped according todose of snuff (naswar) per day, it was observedthat maximum number of patients (n = 45) fellin Group 2, followed by (n = 39) in Group 3,while the least number of cases (n = 16) werewhile the least number of cases (n = 16) werefound in Group 1.

• Likewise when the dose of snuff per day wasassociated with the clinical features, we notedthe following results

* Group 1 = Mild ( less then 5 times a day )* Group 2 = Moderate ( Between 5 to 10 times a day )* Group 3 = Heavy ( More then 10 times a day )

Group 1n (%)

Group 2n (%)

Group 3n (%)

Total P Value

MUCOSITIS 16 (100 ) 45 (100 ) 39 (100 ) 100 (100) --

ULCERATION 16 (100 ) 45 (100 ) 39 (100) 100 (100) --

WHITE PLAQUE 6 (38) 10 (22.2) 15 (38.5) 31 (31 ) 0.257

XEROSTOMIA 16 (100 ) 45 (100 ) 39 (100 ) 100 (100 ) --

ERYTHROPLAKIA 5 (31.2) 37 (82) 31 (80) 73 (73 ) 0.001*

* Fishers exact test

**Pearson Chi square test

* Group 1 = Mild ( less then 5 times a day )

* Group 2 = Moderate ( Between 5 to 10 times a day )

* Group 3 = Heavy ( More then 10 times a day )

PERIODONTITIS 11 (69 ) 17 (39) 7 (18) 35 (35 ) 0.001**

TOBACCO POUCH KERATOSIS

16 (100 ) 45 (100 ) 39 (100 ) 100 (100 ) --

GINGIVITS 10 (62.5) 28 (62.2) 24 (62 ) 62 (62 ) 0.997

CYTOPATHOLOGICAL OBSERVATIONS

• Microscopic examination revealed variety of orallesions ranging from simple inflammation toatypical as well as dysplastic transformations.atypical as well as dysplastic transformations.

CYTOLOGICAL CHANGES SEEN IN n = 100

PATIENTS

36%

26%

3%3% percentage

Inflammation 42 % (41 % acute, 1 % chronic

32%

26%Nuclear atypia 37 %

Micronuclei 30 %

candida 4 %

Dysplasia 4 %

Inflammatory Atypia (H/E)

Inflammatory Atypia – (PAP)

Inflammatory Atypia – moderate degree (H/E)

Epithelial Atypia (H/E)

Epithelial Atypia (PAP)

Epithelial Atypia (H/E)

Pleomorphic clump of epithelial cells (PAP)

Grade 1 dysplasia

Grade 1 dysplasia

Grade 1 dysplasia

Grade 2 dysplasia (H/E)

Grade 2 Dysplasia (H/E)

Spores of Candida (PAS)

Spores of Candida (GMS)

Spores of Candida (GMS)

Spores of Candida (GMS)

• As regards inflammation n = 26 (62 %) smearswith mild, n = 7 (16 %) with moderate while n =9 (22 %) smears with severe degree ofinflammatory infiltrate were seeninflammatory infiltrate were seen

Inflammatory Atypia – mild degree (H/E)

Inflammatory Atypia – mild degree (PAP)

Inflammatory Atypia – moderate degree (H/E)

Inflammatory Atypia – moderate degree (PAP)

Smear showing severe acute inflammatory infiltrate with degenerating sq. epithelial

cells (arrows)

• When these cytopathological changes wereassociated with the age groups followingassociations were observed.

Group 1n (%)

Group 2n (%)

Total P value

INFLAMMATION 18 (42) 23 (40.4) 41 (41 ) 0.879

NUCLEARATYPIA

18 ( 42) 19 (33.3 ) 37 (37 ) 0.382

MICRONUCLEI 5 (11.6) 4 (7.0) 9 (9 ) 0.493

Group 1 = age group less then 30 years

Group 2 = age group above 30 years

CANDIDA 1 3 4 (4 )

DYSPLASIA 1 (2.3 ) 3 (5.3) 4 (4) 0.632

CYTOPLASMIC VACUOLIZATION

13 (30.2) 24 (42.1) 37 (37 ) 0.223

• When these cytopathological changes wereassociated with duration of snuff, the followingassociations were seen

Group 1n (%)

Group 2n (%)

Group 3n (%)

Total P Value

INFLAMMATION 18 (33.3 ) 21 (51.2) 2 (40 ) 41 (41 ) 0.247

NUCLEARATYPIA

9 (17) 25 (61 ) 3 (60) 37 (37 ) 0.000*

MICRONUCLEI 5 (9.3) 3 (7.3) 1 (20 ) 9 (9) 0.562

CANDIDA 1 2 1 4 (4)

* FISHERS EXACT TEST

** FISHERS EXACT TEST

Group 1 = mild � less then 5 years of intake

Group 2 = moderate � between 5 to 10 years of usage

Group 3 = heavy � more then 10 years of usage

CANDIDA 1 2 1 4 (4)

DYSPLASIA 1 (2) 2 (5 ) 1 (20) 4 (4) 0.139

CYTOPLASMIC VACUOLIZATION

12 (22.2) 21 (51.2 ) 4 (80 ) 37 (37) 0.001**

• When these cytopathological changes wereassociated with dosage of snuff per day thefollowing observations were made

Group 1n (%)

Group 2n (%)

Group 3n (%)

Total P Value

INFLAMMATION 5 (31.2) 16 (36) 20 ( 51.3 ) 41 (41 ) 0.236

NUCLEARATYPIA

1 (6.2) 13 (29) 23 (59 ) 37 (37 ) 0.000*

MICRONUCLEI 0 (0 ) 3 (7 ) 6 (15.4) 9 (9 ) 0.196

CANDIDA 1 1 2 4 (4)

* PEARSON CHI SQUARE TEST

** FISHERS EXACT TEST

*** PEASRON CHI SQUARE TEST

*Group 1 = Mild ( less then 5 times a day )

*Group 2 = Moderate ( Between 5 to 10 times a day )

*Group 3 = Heavy ( More then 10 times a day )

DYSPLASIA 0 (0 ) 0 (0 ) 4 ( 10.3 ) 4 (4) 0.033**

CYTOPLASMIC VACUOLIZATION

0 (0 ) 12 (27) 25 (64.1 ) 37 (37 ) 0.000***

HIGHLIGHTS OF SIGNIFICANT ASSOCIATIONS

• Erythropakia when associated with dose of snuff

per day (p=0.001)

• Periodontitis when associated with dose of snuff• Periodontitis when associated with dose of snuff

per day (p=0.001)• P=0.021 (Yong Chu et al, 2010; Columbia)

• P=0.002 (Bergstrong, 2006; Sweden)

• P=0.001 (Anand et al, 2013; India)

• Monten et. al, 2006; Sweden. No. significant association of dose and periodontitis.

• Similarly when duration of snuff usage was

associated with nuclear atypia the value of p

came out significant p = 0.000

• Cytoplasmic vacuolization associated with

duration of snuff usage also showed a significant

p value of 0.001

• Cytoplasmic vacuolization, nuclear atypia and

dysplasia associated with dosage of snuff per day

came out significant with p values 0.000, 0.000

and 0.033 respectively.• Hirsch et. al., 1982; Sweden and Peterson et al, 1983; Denmark, associated dysplasia with duration of snuff usage• Hirsch et. al., 1982; Sweden and Peterson et al, 1983; Denmark, associated dysplasia with duration of snuff usage

• Greer et. al., ( USA ) reported 23 % Atypia and 2 %Dysplasia

• Peterson et. al., ( Danish study ) reported 3%Dysplsia

INCREASING TRENDS OF CLINICAL LESIONS WITH AGE,

DURATION AND DOSAGE OF NASWAR

• White plaque, erythroplakia, xerostomia,tobacco pouch keratosis and gingivitis werefound to be an increasing trend in patients whowere of age group 2.found to be an increasing trend in patients whowere of age group 2.

• Similarly mucositis, ulceration, white plaque,erythroplakia, xerostomia, periodontitis,gingivitis and tobacco pouch keratosis, all werefound to be increasing with the increase in thedose of snuff per day ranging (Group 2 to Group3)

INCREASING TRENDS OF CYTOPATHOLOGICAL

CHANGES WITH AGE, DOSE AND DURATION OF SNUFF

• Inflammation, nuclear atypia, vacuolizationand dysplasia were seen to be more prominentcytopathological features in patients above thecytopathological features in patients above theage of 30 (Group 2).

• Similarly all the above cytopathologicalfeatures were seen to be gradually increasingwhen the dosage per day was increased (Group2 to Group 3).

DISCUSSION

Clinical Variables Total Studies in Pakistan International studies

Age 42.5 + 13.8 39.2 mean age(Zahoor rana et al, 2009)

16 to 24 years(Rolandson et al, 2006; sweden)13.4 years (offenbacher et al, 1985; Southafrica)36.5 years(Wickholm, 2003; Switzerland)

Socioecomic status Low 97 % Low(Zahoor rana et al , 2009)

Mostly Low (Frithiof et al, 1983; Sweden)(Christensen et al, 1979; USA)

Mucositis 12 % -- 46%(Robertson, 1990; Canada)75%(Rolandson et al, 2006; sweden)

Clinical Variables Total Studies in Pakistan International studies

Gingivitis 62 % -- 47%(Monten et al, 2006; Sweden)12 %(Rolandson et al, 2006; Sweden)Insignificant Offenbacher, 1985; Southafrica

Periodontitis 35 % -- 32%Periodontitis 35 % -- 32%(malagi et al, 2013; India)19%(rolandson, 2006; Sweden)36 %(yong chu, 2010; Columbia)

Erythroplakia 73 % ---------

White lesion 31 % 50%(Zahoor rana et al, 2009)

59%(monten et al, 2006; Sweden)36%(Malagi, 2013; India)

CytopathologicalVariables

Total Studies in Pakistan International studies

InflammationMildModerateSevere

42 %62 %16 %22 %

-------- 76 % mild14 % mod9 % severe(Axel et al, 1976; Sweden)46%(Robertson, 1990; Canada)

Nuclear atypia 37 % --------- 23%(Greer et al, 1986; USA)18%18%(erenmemisoqlu, 2007; Turkey)

Dysplasia 4 %(G1=50 %G2=50 %)

12 %(Zahoor rana, 2009)

1%(Peterson et al, 1973; Denmark)2.2%(Greer et al, 1986; USA)4%(G1=75 %. G2=25%)(Erenmemisoqlu, 2007; Turkey)

Cytopathological changes

Total Studies in Pakistan International studies

Candida 4 % -- -----

Cytoplasmic vacuolization 37 % 26 % (Axet et al, 1976; Sweden)

Micronuclei -- 60%(palaskar, 2009; India)74%(roberts et al, 2014; USA)

CONCLUSION

• This study is likely to help in screening habitual dippingtobacco users for pathological changes in oral cavityranging from benign to precancerous and cancerouslesions.

• This study helped in creating awareness among thisindigent population regarding oral and dental health inorder to provide adequate and timely preventive as wellas curative measures by the dental practitioners. As earlydetection of such mucosal changes will improve theclinical outcome.

• To validate the findings of this study, similar

studies on larger scale should be carried out.

• There is a dire need to draft and enforce national

policy and regulations for snuff usage and its

manufacturing creating provisions of adequately

tested material.

Folkman J. Tumor angiogenesis. In: Cancer

Medicine 2000;5th ed:13-52